Neural stem cells and use thereof for brain tumor therapy

ABSTRACT

The present invention is based upon a surprising finding that stem cells, more particularly neural stem cells, can migrate throughout a brain tumor and track metastatic brain tumor cells. The invention provides a method for treating brain tumors by administering genetically engineered neural stem cells in an individual affected by brain tumors. The invention also provides a method of preparing genetically engineered neural stem cells and a composition comprising genetically engineered neural stem cells in a pharmaceutically acceptable carrier.

GOVERNMENT SUPPORT

This invention was made in part with support from the NationalInstitutes of Health under grant number NIH P20-HD18655, and the UnitedStates government has certain rights in this invention.

RELATED APPLICATIONS

This application claims priority of a provisional application 60/185,572filed on Feb. 28, 2000 and is a continuation-in-part of a pending U.S.application, Ser. No. 09/168,350, filed on Oct. 7, 1998, which is acontinuation-in-part of pending U.S. application Ser. No. 09/133,873,filed on Aug. 14, 1998, which applications are incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is in the field of gene therapy, more particularly thefield of using neuronal cells to treat brain tumors. The presentinvention further relates to the field of genetic engineering andmedical treatment with genetically engineered stem cells. Moreparticularly, the invention relates to a method of treatment of CNStumors using genetically engineered neural stem cells (NSCs).

2. Technical Background

An effective gene therapy for the treatment of brain tumors has been anelusive goal for many years. Glioblastoma multiforma, which is virtuallyuntreatable, and the less malignant anaplastic astrocytoma account forabout one-quarter of the 5,000 intracranial gliomas diagnosed yearly inthe United States; 75 percent of gliomas in adults are of this category.Because of its profound and uniform morbidity, it contributes more tothe cost of cancer on a per capita basis than does any other tumor. Thepatient, commonly stricken in the fifth decade of life, enters a cycleof repetitive hospitalizations and operations while experiencing theprogressive complications associated with relatively ineffectivetreatments of radiation and chemotherapy [“Harrison's Principles ofInternal Medicine,” edited by Issetbacher, Braunwald, Wilson, Martin,Fauci and Kasper, 13th Edition, p. 2262, McGraw-Hill, Inc. 1994].

One of the impediments to gene therapy of brain tumors such as gliomas,has been the degree to which they expand, migrate widely and infiltratenormal tissue. Most gene therapy strategies to date are viralvector-based, yet extensive distributions of sufficient amounts of viralvector-mediated genes to large regions and numbers of cells typically inneed has often been disappointingly limited. Interestingly, one of thedefining features of normal neural progenitors and stem cells is theirmigratory quality. Neural stem cells (NSCs) are immature, uncommittedcells that exist in the developing, and even adult, CNS and postulatedto give rise to the array of more specialized cells of the CNS. They areoperationally defined by their ability to self-renew and todifferentiate into cells of most (if not all) neuronal and gliallineages in multiple anatomical & development contexts, and to populatedeveloping and/or degenerating CNS regions [Ciage et al., Ann RevNeurosci 18: 159-92, 1995; Whittemore et al., Molecular Neurobiology12:13-39 1996; McKay Science 276: 66-71, 1997; Gage F H, Christen Y.(eds.), Research & Perspecti'ves in Nourotciences: Isolation,Characterization, & Utilization of CNS Stem Cells, Springer-Verlag,Heidelberg, Berlin, 1997; Snyder, The Neuroscientist 4, 408-25, 1998].

With the first recognition that neural cells with stem cell properties,reproduced in culture, could be reimplanted into mammalian brain wherethey could reintegrate appropriately and seamlessly in the neuralarchitecture and stably express foreign genes gene therapists began tospeculate how such a phenomenon might be harnessed for therapeuticpurposes [Snyder et al., Cell 68: 33-51 1992; Renfranz et al., Cell 66:713-729, 1991]. These, and the studies which they spawned, provided hopethat the use of neural progenitor/stem cells, by virtue of theirinherent biology, might circumvent some of the present limitations ofpresently available gene transfer vehicles (e.g., non-neural cells,viral vectors, synthetic pumps), and provide the basis for a variety ofnovel therapeutic strategies [for review, see e.g., [Ciage et al., AnnRev Neurosci 18: 159-92, 1995; Whittemore et al., Molecular Neurobiology12:13-39 1996; McKay Science 276: 66-71, 1997; Gage F H, Christen Y.(eds.), Research & Perspecti'ves in Nourotciences: Isolation,Characterization, & Utilization of CNS Stem Cells, Springer-Verlag,Heidelberg, Berlin, 1997; Snyder, The Neuroscientist 4: 408-25, 1998;Snyder et al., Current Opin in Pediatrics 8: 558-568, 1996].

The use of neural stem cells as graft material has been clearlyillustrated by the prototypical neural progenitor clone, C17.2, a clonewith which we have had extensive experience which was used in thestudies presented here [Snyder et al., Cell 68: 33-51 1992; Snyder etal., Nature 374: 367-370, 1995; Park, J Neurotrauma 16: 675-87, 1999;Aboody-Guterman et al., NeuroReport 8: 3801-08, 1997]. C17.2 is a mousecell line from postnatal day 0 cerebellum immortalized by infection witha retroviral construct containing the avian myc gene. This line has beentransduced to constitutively express the lacZ and neoR genes. Whentransplanted into germinal zones throughout the brain, these cells havebeen shown to migrate, cease dividing, and participate in the normaldevelopment of multiple regions at multiple stages (fetus to adult)along the murine neuraxis, differentiating appropriately into diverseneuronal and glial cell types as normal, nontumorigeniccytoarchitectural constituents. They intermingle non-disruptively withendogenous neural progenitor/stem cells, responding to the same spatialand temporal cues in a similar manner. Crucial for therapeuticconsiderations, the structures to which C17.2 cells contribute developand maintain neuroanatomical normality. In their earliest therapeuticuse, they served to deliver a missing gene product throughout the brainsof mice with a lysosomal deficiency state and cross-corrected host cellsby release and uptake of a lysosomal enzyme [Snyder et al., Nature 374:367-370, 1995]. The feasibility of a neural progenitor/stem cell-basedstrategy for the delivery of therapeutic molecules directly to andthroughout the CNS was first affirmed by correcting the widespreadneuropathology of a murine model of the genetic neurodegenerativelysosomal storage disease mucopolysaccaridosis type VII, caused by aninherited deletion of the β-glucuronidase (GUSB) gene, a condition thatcauses mental retardation and early death in humans. Exploiting theirability to engraft diffusely and become integral members of structuresthroughout the host CNS, GUSB-secreting NSCs were introduced at birthinto subventricular germinal zone, and provided correction of lysosomalstorage in neurons and glia throughout mutant brains. In so doing, itestablished that neural transplantation of neural progenitor cells couldprovide a novel therapeutic modality.

What is needed is a way to treat tumors which are diffuse, infiltratingand/or metastasizing. What is needed is a way to treat tumors locally tomaximize the impact on the tumor and reduce the toxicity to the patient.

SUMMARY OF THE INVENTION

An isolated pluripotent neuronal cell having the capacity todifferentiate into at least different types of nerve cells is disclosed.The pluripotent cell is further characterized by having a migratorycapacity whereby the cell is capable of traveling from a first locationwhere the neuronal cell is administered to a second location at whichthere is at least one tumor cell, having the ability to travel throughand around a tumor, whereby a plurality of the neuronal cells arecapable of surrounding the tumor; and having the capacity to track atleast one infiltrating tumor cell, thereby treating infiltrating andmetastasizing tumors.

The neuronal cell may be an isolated neural stem cell. The neuronal cellcan be genetically engineered to secrete a cytotoxic substance. In oneembodiment, the neuronal stem cell is genetically engineered using aviral vector encoding a therapeutic gene. In another embodiment theneuronal cell can be genetically engineered to express a suicide gene, adifferentiating agent, or a receptor to any number of trophins. Theneuronal cells if administered on the same side or a contralateral sideof the brain from the tumor, are capable of reaching the tumor cells.

In another embodiment there is provided a method of converting amigrating neuronal cell to a migrating packaging/producer cell, saidmethod includes the steps of a) providing a neuronal cell whichconstitutively produces a marker such as β-gal; b) cotransfecting theneuronal cell with an amphotropic pPAM3 packaging plasmid and apuromycin selection plasmid pPGKpuro; c) selecting transfected cells inpuromycin; d) selecting for cell surface expression of the amphotropicenvelope glycoprotein coat; e) isolating cells by fluorescent activatedcell sorting using monoclonal antibody 83A25; and f) screening the cellsof step e for their packaging ability by assessing which coloniespackaged lacZ into infectious viral particles. Thus there is produced amigratory neuronal cell capable of being transfected with a gene ofchoice, so that viral particles expressing the gene of choice areproduced and disseminated over a wide area of the central nervous systemby a plurality of the transfected packaging cells.

The method of converting the migratory neuronal cell into a packagingcell line wherein step f. is performed by a virus focus assay for β-galproduction. Alternatively the method can be performed with a prodrugactivation enzyme as the gene of choice. Alternatively, the, prodrugactivation enzyme is E. coli cytosine deaminase (CD), HSV-TK orcytochrome p450. More preferably, the prodrug activation enzyme is E.coli cytosine deaminase (CD).

Also disclosed is a novel cell packaging line for the central nervoussystem. The cell line includes neuronal cells which constitutivelyproduce a marker such as 0-gal, have been cotransfected with anamphotropic pPAM3 packaging plasmid and a puromycin selection plasmidpPGKpuro; are selected in puromycin, for cell surface expression of theamphotropic envelope glycoprotein coat and for fluorescence usingmonoclonal antibody 83A25, and for their packaging ability by assessingwhich colonies packaged lacZ into infectious viral particles. Theresulting cells are capable of packaging and releasing particles orvectors which, in turn, may serve as vectors for gene transfer tocentral nervous system cells. The particles in the novel cell packagingline can be replication-defective retroviral particles. The vectors inthe novel cell packaging line can be replication-conditional herpesvirus vectors.

The present invention is based upon a surprising finding that stemcells, more particularly neural stem cells, when administeredintracranially can migrate throughout a tumor and track metastatic tumorcells to reach tumor cells in the brain. The invention provides a methodfor treating brain tumors by administering genetically engineered stemcells, more preferably neural stem cells in an individual affected withbrain tumors. The invention also provides a method of preparinggenetically engineered neural stem cells and a composition comprisingthe genetically engineered stem cells in a pharmaceutically acceptablecarrier.

It is a further object of this invention to provide a safe, efficientand convenient system for delivering therapeutic agents to intracerebraltumors, cerebral metastases from an extracerebral tumor.

In one embodiment, the present invention provides a neuronal stem cellcomprising a vector encoding a therapeutic agent. In one embodiment thevector is a replication conditional vector. In a preferred embodimentvector is a herpes simplex vector and in a most preferred embodiment thevector is a herpes simples type 1 vector. In a further preferredembodiment the herpes simplex type 1 vector is deficient forribonucleotide reductase.

The invention also provides a method of treating a brain tumor in amammal in need thereof, the method comprising a) providing a neuronalstem cell comprising a vector encoding a therapeutic agent; and b)administering said neuronal stem cell in a pharmaceutically acceptablecarrier into a mammal in need thereof. In one embodiment, the method isused for treating a malignant glioma.

In one embodiment, replication of the vector is controlled by making thevector deficient for a component necessary for vector replication. In apreferred embodiment, the vector is made deficient for ribonucleotidereductase.

The invention also provides a method of treating a brain tumor in amammal in need thereof the method comprising: a) providing a neuronalstem cell comprising a replication conditional vector encoding atherapeutic agent; b) inhibiting replication of said replicationconditional vector in said neuronal stem cell; c) administering theneuronal stem cell of step b in a pharmaceutically acceptable carrierinto a mammal in need thereof; and, d) enhancing replication of saidreplication conditional vector. In one embodiment, step b) is performedby inhibiting growth of the neuronal stem cell. In a preferredembodiment the growth inhibition is performed using mimosine. In afurther embodiment, growth inhibition is performed using a combinationof mimosine and ganciclovir.

The present invention further provides a method of treating a braintumor in a mammal in need thereof said method comprising: a)administering into a mammal a neuronal stem cell comprising a herpessimplex type 1 vector encoding thymidine kinase; and b) administeringganciclovir into said mammal.

The invention also provides a method of preparing neural stem cellsencoding a therapeutic agent, said method comprising: a) providing aneural stem cell; b) growing said neural stem cell to confluency; c)subjecting the neural stem cell to a replication-arresting protocol; d)infecting the replication arrested cell with RR-P450; and e) washing theinfected cell, separating the cell from its growth surface andresuspending the cell in a medium to obtain a concentration of 50,000cells/μl.

In a preferred embodiment, a method of preparing neural stem cellsencoding a therapeutic agent comprises: a) providing a neural stem cell;b) growing said neural stem cell to confluency; c) subjecting the neuralstem cell to a replication-arresting protocol, said protocol comprisingtreating cells with a medium comprising about 400 μM mimosine on days 0and 4 and treating cells on day 6 with a medium comprising about 400 μMmimosine and optionally about 5 μM ganciclovir; d) infecting thereplication arrested cell with RR-P450 at an MOI of 1 on day 7; and e)washing the infected cell, trypsinizing and resuspending in DMEM andoptionally 5 μM GCV to obtain a concentration of 50,000 cells/μl.

BRIEF DESCRIPTION OF FIGURES

FIGS. 1A and 1B illustrate the migratory capacity of neuralprogenitor/stem C17.2 cells in vitro. After 5 days of incubation therewas a wide distribution of C17.2 cells (FIG. 1B), suggesting that theyhad migrated far from their initial seeding in the cylinder, compared toTR-10 cells (FIG. 1A), which remained localized to the area of initialseeding in the cylinders. These patterns were observed whether the cellswere plated directly on top of the glioma cells (right-sided cylinder(arrows) or simply in juxtaposition to them (center cylinder arrows).

FIGS. 2A, 2B, 2C and 2D illustrate foreign gene-expressing neuralprogenitor/stem cells extensive migration throughout experimental tumormass, and slightly beyond advancing tumor edge, appearing to “track”migrating tumor cells. (FIG. 2A) day 2 shown at 4X; arrowheads demarcatethe approximate edges of tumor mass; (FIG. 2B) high power at 10X whereX-gal, blue-staining NSCs (arrows) are interspersed between tumor cellsstaining dark red. (FIG. 2C) View of tumor mass 10 days afterintratumoral injection showing X-gal-blue, C17.2 NSCs have infiltratedthe tumor but largely stop at the edge of the darkly red stained tumortissue with some migration into surrounding tissue when theblue-staining NSC appears to be “following” an invading, “escaping” cell(arrow) (10X). (FIG. 2D) CNS-1 tumor cells implanted into an adult nudemouse frontal cortex, there is extensive migration and distribution ofblue C17.2 cells throughout the infiltrating experimental tumor bed, upto and along the infiltrating tumor edge (arrows), and where many tumorcells are invading normal tissue, into surrounding tissue in virtualjuxtaposition to aggressive tumor cells (arrows) (10X).

FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G and 3H illustrate the neuralprogenitor/stem cel appearance to “track” migrating tumor cells awayfrom main tumor mass, (FIGS. 3A, 3B) parallel sections: low power C17.2cells distributed throughout tumor and surrounding edge (FIG. 3A) X-galand neutral red, FIG. 3B) double immunofluorescent labeling with TexasRed and FITC; (FIGS. 3C, 3D) low and high power of tumor edge andmigrating tumor cell in juxtaposition to C17.2 cell (X-gal and neutralred); (FIGS. 3G, 3H) low and high power of single migrating tumor cellsin juxtaposition to C17.2 cells (double immunofluorescent labeling withTexas Red and FITC).

FIGS. 4A, 4B, 4C, 4D, 4E, 4F and 4G illustrate neural progenitor/stemcells implanted at distant site from main tumor bed migrating throughoutnormal tissue target CNS-1 tumor cells; (FIGS. 4A, 4B) same hemisphere:3×10⁴ CNS-1 tumor cells implanted into right frontal lobe. On day 6,4×10⁴ C17.2 cells injected into fight frontoparietal lobe (4 mm caudaltumor injection). Animals sacrificed on day 12 (shown) and day 21, C17.2cells seen in tumor bed (X-gal and neutral red). (FIGS. 4C, 4D, 4E)Contralateral hemisphere: 3×10⁴ CNS-1 tumor cells implanted into leftfrontal lobe and 5×10⁴ CNS-1 tumor cells implanted into leftfrontoparietal lobe. On day 6, 8×10⁴ C17.2 cells were injected intofight front lobe. Animals were sacrificed on day 12 and 21 (shown); c)4×C17.2 cells (red) seen migrating towards tumor (green) from oppositeside of the brain, d) 10X C17.2 cells (red) seen actively migratingacross central commisure (double immunofluorescence), e) 20×C17.2 cells(blue) seen entering tumor (black arrows) (X-gal/Neutral Red). FIGS. 4F,4G show intraventricular 5×10⁴ CNS-1 tumor cells were implanted intoright frontal lobe. On day 6, 8×10⁴ C17.2 cells were injected into rightor left (shown) lateral ventricle.

FIG. 5 shows the schedule of drug treatment and superinfection of neuralstem cells with ribonucleotide reductase-negative HSC-1 mutants. Earlypassages of neural stem cells in log-growth phase were gown toconfluency (400,000 cells per well in 24-well dish), treated with 400 μMmimosine and 5 μM ganciclovir (GCV), and infected with hrR3 or RRP450 atan MOI of 1. Abbreviations: DMEM is Dulbecco's modified Eagle's medium.FCS is fetal calf serum and RR is ribonucleotide reductase.

FIG. 6 shows a growth curve of uninfected neural stem cells followingtreatment with mimosine alone or mimosine and GCV and/or infection withreplication-conditional virus. Abbreviations: Mim means cells treatedwith 400 μM mimosine; MG means cells treated with 400 μM mimosine and 5μM GCV; split means cell cultures split 1:8 on day 10.

FIG. 7 shows a growth curve of neural stem cells following treatmentwith mimosine alone or mimosine and GCV which were infected with RRP450replication-conditional virus at a MOI 1 on day 7.

DETAILED DESCRIPTION OF THE INVENTION

The experiments presented herein demonstrate that NSCs (prototypicalclone C17.2) when implanted into an experimental glioma, will distributethroughout the tumor and migrate along with aggressively advancing tumorcells, while continuing to express their reporter gene lacZ. (One of theglioma lines used, astrocytoma cell line CNS-1, demonstrates single cellinfiltration and invasive characteristics similar to those of humanglioblastomas”). Furthermore, the neural progenitor/stern cells seem tomigrate slightly beyond and surround the invading tumor border. Inadditional experiments, where neural progenitors were implanted at adistant site from the tumor bed, in the same hemisphere, oppositehemisphere, or lateral ventricle, they migrated through normal tissuemoving specifically toward CNS-1 tumor cells. They were found toaccumulate in or near the tumor bed as well as near or in directjuxtaposition to the individual infiltrating tumor cells.

Not wishing to be bound by any particular theory, the inventors proposethat this neural progenitor/stem cell system migrate towards a trophicgradient of growth factors produced by the tumor cells. Thus, NSCs mayprovide a unique platform for the dissemination of therapeutic genes tothe proximity of or into tumors that previously were inaccessible. Theseobservations further suggest a number of other new gene therapyapproaches. These may include the dissemination of cytotoxic geneproducts, but could also include factors that directly promotedifferentiation of neoplastic cells as well as the more efficaciousdelivery of viral vectors encoding therapeutic genes to be incorporatedby tumor cells (e.g. suicide genes, differentiating agents, receptors totrophins). Because NSCs can be engineered to package and releasereplication-defective retroviral particles or replication-conditionalherpes virus vectors which, in turn, may serve as vectors for thetransfer of genes to CNS cells, neural progenitor/stem cells shouldserve to magnify the efficacy of viral-mediated gene delivery to largeregions in the brain.

One effective mode of therapy for experimental brain tumors has beenprodrug activation. Initially, prodrug activation enzymes were limitedto antibodies directed against tumor enriched antigens. New strategiesincorporate genes for these enzymes into viral vectors. Among theprodrug activating systems shown to be effective for gliomas E. colicytosine deaminase (CD), HSV-TK and cytochrome p450 have beendemonstrated to have a drug mediated bystander effect. Of these CD givesthe best reported “bystander” effect. CD converts the nontoxic prodrug5-fluorocytosine (5-FC) to 5fluorouridine (5-FU) metabolites. 5-FU is achemotherapeutic agent which has selective toxicity for activelydividing cells, thus primarily targeting tumor cells. In addition, 5-FUand its toxic metabolites can readily pass into adjacent and surroundingcells by nonfacilitated diffusion. Brain tumors may require only a smallnumber of cells expressing CD (about 2% evenly distributed) to generatesignificant anti-tumor effects when treated with systemic, non-toxiclevels of 5-FC. Our results support the hypothesis that transduced NSCswould disperse CD expression efficiently throughout the tumor and even“track” single migrating, “escaping” tumor cells.

Another approach to brain tumor gene therapy has been selective genetransfer to tumor cells in combination with pharmacotherapy, e.g., theHSV-TK gene, when transduced via retrovirus into a dividing populationof brain tumor cells, confers a lethal sensitivity to the drugganciclovir. Recent modifications of retroviral constructs to increaseefficiency of infection and cell-specific targeting hold promise forenhancing the potency of this strategy. Again, through the “bystander”effect, tumor destruction is effective even when only a fraction of thecells express HSV-TK; adjacent tumor cells not expressing HSV-TK alsoappear to be eliminated. Attempts to improve efficiency of tumordestruction have focused on increasing the number of cells expressingthe HSV-TK gene. The use of NSCs as packaging cells (which might then beself-eliminated) may prove to be an effective extended delivery systemof the lethal gene to neighboring mitotic tumor cells, especiallyindividual, infiltrating tumor cells.

In conclusion, genetically modified neural progenitor/stem cells havethe potential to supply a range of tumor selective agents throughoutmature and developing brains. The experiments presented here demonstratethe ability of NSCs: (1) to migrate/distribute quickly and effectivelythroughout the main tumor bed when implanted directly into theexperimental gliomas; (2) to migrate slightly beyond and “surround” (asif to contain) the invading tumor border; (3) to seemingly “track”individual, infiltrating tumor cells into surrounding tissue; (4) tomigrate through normal tissue from distant sites to target CNS-tumors;and (5) to show stable expression of a foreign gene, in this case lacZ,throughout the tumor bed and in juxtaposition to tumor cells. Theseresults lay the groundwork for future therapeutic brain tumor studies,providing critical support for the use of neural progenitor/stem cellsas an effective delivery vehicle for tumor directed, vector-mediatedenzyme/prodrug gene therapy.

Other cells useful according to the invention include, but are notlimited to the HCN-1 cell line which is derived from parental cell linesfrom the cortical tissue of a patients with unilateral megalencephalygrowth [Ronnett G. V. et al., Science 248: 603-5, 1990]. HCN-1A cellshave been induced to differentiate to a neuronal-like morphology andstain positively for neurofilament, neuron-specific enolase and p75NGFR,but not for myelin basic protein, S-100 or glial fibrillary acidicprotein (GFAP). Because these cells also stain positively for 7-aminobutyric acid and glutamate, they appear to become neuro-transmittingbodies. Earlier, it has been observed that HCN-I cells survived in thebrain parenchyma and proposed that these cells may be suitable forintracerebral transplantation in humans [Poltorak et al., CellTransplant 1: 3-15, 1992].

It has also been reported that HCN-1 cells grew processes resemblingneurons when exposed to nerve growth factor, dibutyryl cyclic AMP andisobutylmethylxanthine [Ronnet et al., Neuroscience 63: 1081-99, 1994].

The nerve cells also can be administered with macrophages which havebeen activated by exposure to peripheral nerve cells. Such activatedmacrophages have been shown to clean up the site of CNS trauma, forexample a severed optic nerve, after which new nerve extensions startedto grow across the lesion. Implanting macrophages exposed to CNS tissue(which secretes a chemical to inhibit macrophages) or nothing at allresulted in little or no regeneration [Lazarov-Spiegler et al., FASEB J10: 1, 1996].

Fetal pig cells have been implanted into patients with neurodegenerativediseases, such as Parkinson's disease and Huntington's chores, andintractable seizures, in whom surgical removal of the excited area wouldotherwise have been performed. Such cells, if properly screened forretroviruses, could also be used in the Inventive method.

Neural crest cells can be isolated and cultured, e.g. according toStemple and Anderson (U.S. Pat. No. 5,654,183), which is incorporatedherein by reference, with the modification that basic fibroblast growthfactor (bFGF) is added to the medium at concentration ranging from 5-100ng/ml in 5 ng/ml increments. Neural crest cells so cultured are found tobe stimulated by the presence of FGF in increasing concentrations about1 or 5 ng/ml. Such cells differentiate into peripheral nerve cells,which can be used in the instant invention.

The invention further provides a method for treating tumors byadministering neural stem cell comprising a vector encoding atherapeutic agent in an individual affected by a brain tumor. Theinvention also provides a method of preparing the neural stem cells anda composition comprising the genetically engineered stem cells in apharmaceutically acceptable carrier.

The genetically engineered neural stem cell based delivery method of thepresent invention offers a number of advantages over direct injection ofvirus into a tumor. For example, the virus can be activated after adelay to allow the cells to migrate towards metastatic tumor cells asdescribed infra.

The neural stem cells useful according to the present invention includecells that are capable of migrating through a tumor, beyond atumor/parenchyma border and brain tissue. These migrating stem cells canbe prepared as described by Snyder [Snyder et al. Cell 68, 33-51, 1992;Snyder, The Neuroscientist 4, 408-25, 1998]. Other examples of migratingstem cells useful according to the present invention include, but arenot limited to, neural stem cells, HSN-1 cells, fetal pig cells andneural crest cells, bone marrow derived neural stem cells, and hNTcells. The HSN-1 cells useful according to the invention can be preparedas described in, e.g., Ronnett et al. [Science 248, 603-605, 1990]. Thepreparation of neural crest cells in described in U.S. Pat. No.5,654,183. The hNT cells useful according to the present invention canbe prepared as described in, e.g, Konubu et al. [Cell Transplant 7,549-558, 1998].

The stem cells according to the present invention are geneticallyengineered to deliver a therapeutic agent that can be used tosubstantially inhibit tumor cell growth. The term “inhibit” as usedherein means inhibiting cell division and growth as well as causingnecrotic or apoptotic cell death.

Such therapeutic agents include, but are not limited to vectors encodinggenes for toxins; prodrugs; enzymes such as cytosine deaminase (CD);angiogenesis inhibitors such as TNP-470, platelet factor 4,thrombospondin-1, tissue inhibitors of metalloproteinases (TIMP1 andTIMP2), prolactin (16-kD fragment), angiostatin (38-kD fragment ofplasminogen), endostatin, bFGF soluble receptor; cytokines; growthfactors and their inhibitors; interleukins (IL), IL I-IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-I0, and IL-I1; tissue necrosis factors (TNF)TNFα and TNFβ; lymphotoxin (LT); interferons (IFN) IFNα, IFNβ and IFNγ;tissue growth factors (TGF); colony-stimulating factors (CSFs); andnerve growth factor (NGF). In the preferred embodiment, the migratingstem cells are engineered to encode cytocine deaminase, which converts anon-toxic 5-fluorocytosine (5-FC) into a toxic 5-fluorouridine (5-FU).

For example, the examples infra show size reduction in the experimentaltumor models in the CD/5-FC prodrug example, NSCs were able to express abioactive transgene in vivo and to effect a significant anti-tumorresult. 5-FU is a chemotherapeutic agent with selective toxicity todividing cells through its toxic metabolites can readily diffuse intosurrounding tumor cells giving CD an impressive “bystander” effect. Aslittle as 2% of the tumor mass expressing CD can generate a significantantitumor effect in response to 5-FC [Huber et al., Proc Natl Acad SciUSA 91: 8302-8306, 1994].

Vectors useful according to the present invention include, but are notlimited to (a) adenovirus vectors; (b) retrovirus vectors; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picarnovirus vectors; (i) vaccinia virus vectors; and (j) ahelper-dependent or gutless adenovirus. In a preferred embodiment thevirus is a herpes simplex type 1 virus (HSV-1). In a most preferredembodiment, the vector is a replication-dependent HSV-1 vector which hasbeen engineered to lack ribonucleotide reductase activity.

In delivery of HSV-1 vectors, it has to be kept in mind that cells whichreplicate HSV-1 will die in the process. Thus, for a cellular deliverysystem of HSV-1 vectors which relies on host cell migration prior tovirus release, it is essential that viral replication is arrested toallow time for migration of infected cells. Ideally, an HSV-1 mutantwould enter a “quiescent” state [Jamieson et al., J Gen Virol 76:1417-31, 1995] in the carrier cell, and replication would be activatedat a defined time. Since replication-conditional HSV-1 vectors do notpropagate in non-dividing cells, the rationale used in these disclosedexperiments was to suppress virus replication by arresting the growth ofneural stem cell carrier cells prior to infection with areplication-conditional mutant virus. The plant non-protein amino acid,mimosine, found in Leucaena and Mimosa genera [Hylin, Biochem Pharmacol14: 1167-9, 1969], was used to arrest progression through the cell cyclefrom G1 to S-phase [Mosca et al., Mol Cell Biol 12: 4375-83, 1992] andto inhibit cellular and viral ribonucleotide reductase (RR) activity bychelating iron, which is essential for RR function [Dai et al., Virology205: 210-6, 1994].

Mimosine treatment suppresses replication of HSV-1 virus deficient forRR both by decreasing cellular RR activity through growth arrest of thehost cells and direct inhibition of activity. Since the effects ofmimosine are reversible, removal of mimosine should allow the cells tocommence growth and restore the function of cellular RR, thus becomingpermissive for replication of the mutant virus.

As an additional guarantee that virus replication is completelyabrogated upon infection, growth-arrested neural stem cells can also betreated with ganciclovir (GCV). GCV acts as a false nucleoside andinhibits viral DNA synthesis in HSV-1 infected cells. GCV or othersubstrates of HSV-TK have been used in cellular models of HSV-1 latencyvirus in neuronal cells [Wigdahl et al., J Virol 49, 205-14, 1984;Wilcox and Johnson, J Virol 61, 2311-5, 1987], and hence may promote asimilar benign, quiescent state in neural progenitor cells. Thus in thepresent model, the mutant virus infecting growth-arrested neural stemcells should enter a quiescent state, and any residual replication willbe blocked by GCV. In this scenario, it is important to know whether thequiescent HSV-1 genomes can be reactivated into a replicating stateafter an extended period. Studies in culture models of HSV-1 latency inneuronal cells have shown that treatment with glucocorticoids [Halfordet al., J Virol 70, 5051-60, 1996; Hardwicke and Schaffer, J Virol 71,3580-7, 1997] or NGF-deprivation [Wilcox and Johnson, J Virol 61,2311-5, 1987] can trigger reactivation. Several HSV-1 proteins, e.g.infected-cell protein (ICP)4 [Kramer and Coen, J Virol 71, 5878-84,1997], ICPO [Zhu et al., J Virol 64, 4489-98, 1990], VP16 [Sears et al.,J Virol 65, 2929-35, 1991], TK [Jacobson et al., J Virol 67, 5383-93,1993; Wilcox et al., Virology 187, 348-52, 1992] and RR [Chang et al.,Virology 185, 437-40, 1991], have all been implicated in thereactivation process of HSV-1 in neuronal cells.

The stem cells can also be engineered to controllably express thetherapeutic agent. Such controlled expression systems include, but arenot limited to drug/hormone inducible promoters, e.g., tetracycline[Gossen and Bujard, Nucl Acids Res 21, 4411-2, 1993], rapamycin [Riveraet al., Nat Med 2, 1028-32, 1996], and glucocorticoid induciblepromoters [Lu and Federoff, Hum Gene Ther 6, 419-28, 1995]; tetracyclinesilencer system [Freundlieb et al., J Gene Med. 1, 4-12, 1999],particularly combined with a “piggyback” HSV-1 delivery system [Pechanet al., Hum Gene Ther 7, 2003-13,1996].

In the preferred embodiment, a replication-dependent HSV-1 vector isproduced by deleting the ribonucleotide reductase (RR) gene of HSV-1vector to render the vector susceptible to control by externalexpression of RR.

To avoid destruction of delivery cells by viral replication uponimplantation, regulation of expression of genes by viral vectors isdesired. Delayed expression allows better migration of the cellsinfected with a viral vector. It is preferred that the expression can bedelayed for 1-6 days, preferably 3 days after the injection of the cellsto avoid self-destruction of the delivery cells and to allow themigrating stem cells to reach potential metastatic tumor cells. Whenusing the inducible systems in viral vectors, it is important to achievefull-off baseline expression to prevent residual viral replication whichcan result in premature death of migrating stem cells infected with the

In one embodiment, the present invention provides migrating neural stemcells infected with an HSV-1 vector which has been engineered to lackthe RR enzyme thereby rendering it non-replicable in the absence ofexternally produced RR. Because the HSV-1 vector can only replicate individing cells, virus replication can be regulated by regulating celldivision.

Control of replication-conditional HSV-1 vector lacking RR can beachieved, for example, by arresting the carrier cell, i.e. the neuralstem cell growth prior to infection. For example, the drug mimosine canbe used to block growth of neural stem cells at confluency and thusprevent virus replication. In addition to arresting the cell cycle inthe late G1 phase, mimosine also inhibits cellular RR enzyme. Additionof mimosine on infected cells in vivo completely abolishes viralreplication which is resumed after removal of mimosine. The mimosineblock of cell division and viral replication is reversible at treatmenttimes at least up to 13 days.

In another embodiment, co-treatment with ganciclovir (GCV) and mimosineas a viral replication block can be used. After GCV treatment, neuralstem cells differentiate into neurons and harbor the virus in a latentstate. After withdrawal of GCV and mimosine the cells need a high levelof RR to allow the re-entry of the quiescent viral genome of thereplication-conditional HSV-1 RR⁻ onto the replicative cycle.Alternatively, the immediate early virus proteins ICPO or ICP4 that areknown to be important in the HSV-1 re-activation can be used tore-activate the arrested viral replication [Zhu et al., J Virol 64,4489-98, 1990]. In addition, viral replication proteins like ICP4 andCIP27 can also be placed under control of drug/hormone induciblepromoters.

Additional genes can be inserted into the replication-dependent vector.A non-limiting example is CYP2B 1 gene, which is responsible for thebio-activation of prodrugs cyclophosphamide and ifosfamide. Once thepackaging cells have migrated to the appropriate site, the appropriateprodrug can be administered to produce an oncolytic effect. Similarly,not all the components of the tested vector are believed to benecessary. Vector constructs may additionally include a marker gene forpotential histological tracking. Such markers include, but are notlimited to lacZ or genes encoding fluorescent proteins such as greenfluorescent protein, GFP.

The migrating stem cells according to the present invention can beadministered to an individual in a pharmaceutically acceptable carrierintracranially, e.g., at the time of surgical removal of the tumor.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA andimmunology, which are within the skill of the art. Such techniques aredescribed in the literature. [See, for example, MOLECULAR CLONING: ALABORATORY MANUAL, 2d ed. Ed. by Sambrook Fritsch and Maniatis ColdSpring Harbor Laboratory Press. 1989; DNA CLONING: VOLUMES I AND II. Ed.by D. N. Glover, 1985; OLIGONUCLEOTIDE SYNTHESIS. Ed. by M. J. Gait,1984; Mullis et al., U.S. Pat. No. 4,683,195; NUCLEIC ACIDHYBRIDIZATION. Ed. by B. D. Hames and S. J. Higgins, 1984; TRANSCRIPTIONAND TRANSLATION. Ed. by B. D. Hames and S. J. Higgins, 1984; CULTURE OFANIMAL CELLS Ed. by R. I. Freshney, Alan R. Liss, Inc., 1987;IMMOBILIZED CELLS AND ENZYMES, IRL Press, 1986; PRACTICAL GUIDE TOMOLECULAR CLONING, B. Perbal, 1984; GENE TRANSFER VECTORS FOR MAMMALIANCELLS, Ed by J. H. Miller and M. P. Calos, Cold Spring HarborLaboratory, 1987; METHODS IN ENZYMOLOGY: VOLS. 154 AND 155, Ed. by Wu etal.; IMMUNNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY, Ed. by Mayerand Walker, Academic Press, London, 1987; HANDBOOK OF EXPERIMENTALIMMUNOLOGY: VOLS. I-IV, Ed. by D. M. Weir and C. C. Blackwell, 1986;MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1986].

The references cited throughout the specification are hereinincorporated in their entirety. The present invention is furtherillustrated by the following examples and claims. The following examplesare provided to aid in the understanding of the invention and are not tobe construed as a limitation thereof.

EXAMPLES

Materials and Methods

Cells

Neural crest cells were isolated and cultured according to Stemple andAnderson [U.S. Pat. No. 5,654,183], which is incorporated herein byreference. With the approximately 60-70% confluency around a 5 mmcylinder (i.e. free of CNS-1 cells) into which 40,000 C17.2 or TR-10cells were plated overnight. At the same time, 40,000 C17.2 or TR-10cells were placed into a 5 mm cylinder placed directly on top of adheredCNS-1 cells. The next day, cylinders were removed and plates rinsed wellwith PBS to remove any floating cells, media was replaced, and platesincubated for 5 days. Plates were subsequently stained for β-galactosideovernight afler. 5% glutaraldehyde fixation. (Note: both C17.2 and TR-10cells were>90% blue with X-gal staining).

The neural stem cells (NSCs) were from a stable, well established, wellstudied, prototypical multipotent engraftable murine neural stem cellclone transfected with and constitutively expressing the lacZ markergene (clone C17.2) [Snyder et al., Cell 68: 33-51, 1992; Snyder et al.,Nature 374: 367-70, 1995; Snyder et al., Proc Natl Acad Sci USA 94:11663-68, 1997]. Described and characterized extensively elsewhere[Snyder, The Neuroscientist 4: 408-25, 1998], this clone of neural stemcells has been shown to be an effective vehicle for gene transfer to theCNS [Snyder et al., Nature 374: 367-70, 1995; Lacorraza et at., NatureMed 4: 424-29, 1996]. Prototypical human NSC clones H1 and H6 also wereused [Flax et at., Nat Biotechnol 16: 1033-39,1998].

NSCs were transduced with cytosine deaminase. A plasmid using theretroviral pBabePuro backbone [Morgenstern and Land, Nucl Acids Res 18:3 5 87-96, 1990] was constructed to include the E. coli cytosinedeaminase cDNA (1.5 kb fragment) under the LTR promoter (kindly providedby Dr. Michael Black). Retrovirus vectors were packaged byco-transduction of the Cdpuro plasmid with amphotropic (MI2YA) orecotropic (MV 12) envelope-coding plasmid CDNA [Sena-Estees et at, J 15Virol 73: 10426-39, 1999] into 293T/17 cells [Pear et at, Proc Natl AcadSci USA 90: 8392-96, 1993].

Cdpuro retroviral supernatant was harvested as previously described[Sena-Esteves et al., J Virol 73: 10426-39, 1999] and used for multipleinfections of several lacZ-positive NSC clonal lines (human HI, H6 andmurine C17.2). Transduced NSC populations were placed under puromycinselection for at least two weeks.

The CHS-1 rat glioma cell line was generated from a glioma induced in aLewis rat by treatment with N-nitroso-N-methylurea [Kruse et at, JNeuro-Oncology 22: 191-200, 1994] and was obtained from Drs. C. A. Kruseand W. F. Hickey (University of Colorado Health Sciences Center, Denver,Colo.). The CNS-1 cells were engineered via retroviral-mediated genetransduction to constitutively express GFP as previously described[Short et al., J Neurosci Res 27: 427-39, 1990] CHS-1 cells weremaintained in RPMI-1640 (BioWhitaker) supplemented with 10% fetal calfserum (FCS) at 370C in a standard 5% C0₂ incubator at 100% humidity.

In vivo studies: 48 hours prior to transplant, C17.2 and TR-10 cellswere incubated with BUdR (Sigma) at a concentration of 10 μM. Platedcells were rinsed with PBS, trypsinized, resuspended in media andcounted on the Coulter counter. Desired number of cells were spun downat 4° C. in the centrifuge for 4 minutes and 1100 rpm to obtain apellet. Media was removed; cells were rinsed by resuspending in PBS andrespun. PBS was removed and the appropriate amount of PBS added toresuspend cells at final desired concentration. Cells were kept on ice,and gently triturated prior to each animal injection. Cells not labeledwith BUdR were prepared for injection in similar manner.

Animals: Animal studies were performed in accordance with guidelinesissued by the Massachusetts General Hospital Subcommittee on AnimalCare. Animals used: adult CD-Fisher rats (Charles River) and 8-10 weekold adult, approximately 20 gram female nude mice (random bred Swisswhite obtained from Cox 7, MGH-East).

Animals were anesthetized by an i.p. injection of 15 ml of 20% ketamineHCL (KETALAR 100 mg/ml; Parke-Davis, Morris Plains, N.J.), 20% xylazine(ROMPLTN 20 mg/ml; Miles Inc., Shawnee Mission, Kans.), 60% sodiumchloride (0.9%; Abbott Laboratories, North Chicago, Ill.) andimmobilized in stereotactic apparatus (Kopf, Tujunga, Calif.).Intracerebral injections were stereotactically performed by making alinear scalpel skin incision on top of the skull. A burr hole wasdrilled into the skull with a high speed drill 2 mm lateral to thebregma on the coronal suture. After incising the dura with a sterileneedle and obtaining hemostasis, desired number on tumor cells suspendedin 1 μl of 1× Dulbecco's phosphate-buffered salt solution (PBS pH 7.4;Mediatech, Herndon, Va.) were injected with a 26 gauge 5 μl Hamiltonsyringe to specified location (see protocols below) over a 3 to 5 minuteperiod. After retracting the needle over a 2-4 minute period, bone-wax(Ethicon, Somerville, N.J.) was used to occlude the burr hole, betadineapplied to surgical area, and the skin sutured closed. Animals receivinga second injection at a later date were anesthetized, immobilized instereotactic apparatus, and cells injected as per specific protocol (seebelow). Animals were sacrificed on stated days with an overdose ofanesthesia and subsequent intracardiac perfusion with PBS followed by 4%pamfonnaldehyde and 2 mM MgCl₂ (pH 7.4). Brains were removed andpost-fixed overnight at 4° C. and then transferred to 30% sucrose in PBSand 2 mM MgCl₂ (pH 7.4) for 3-7 days to cryoprotect. Brains were storedat −80° C. and then 10-15 micron coronal serial sections were cut tocryostat (Leica CM 3000).

BUdR Labeling of Engrafted C7.2 Cells

Selected animals received 3 intraperitoneal injections of 1 ml/100 gbody weight 20 μM BUDR stock solution (Sigma) over 24 hours prior tosacrifice (0.2 ml/injection per 20 g mouse).

Histopathological and Immunohistochemical Studies

Tissue sections were stained with (1) X-gal and counterstained withneutral red (2) hematoxylin and eosin (3), double immunofluorescentlabeling was performed with Texas Red anti-beta-galactosidase and FITCanti-GFP. Slides were examined with light microscopy, fluorescentmicroscopy. CNS-I tumor cells were also examined without staining underconfocal fluorescent microscopy.

For histological analysis, animals were euthanized on the days specifiedbelow by deep anesthesia with at least 500 μl ketamine/xylazine solutionintraperitoneally (i.p.) and subsequent perfusion with a 4%paraformaldehyde solution in PBS (4% PFA). The brain was removed andpost-fixed in 4% PFA and 2 mM MgCl2 (pH 7.4) for 2 days, cryo-protectedin 30% sucrose in PBS, and frozen at −80° C. Brains were sectioned at10-15 μm using a cryostat and stained with neutral red. For lacZ-encodedβ-galactosidase staining, mounted slices were placed for 24 hours at 37°C. in X-gal staining solution containing 5 mM potassium ferrocyanide, 5mM potassium ferricyanide, 2 mM magnesium chloride, 1 mg/ml X-gal(Fisher, Pittsburgh, Pa.), and 2.5% di-methyl-sulfoxide (Sigma, St.Louis, Mo.) in PBS [Turner et al., Stain Technol 65: 55-67, 1990]. Thesections were washed with PBS and counterstained with hematoxylin.

Cell Lines and Replication-Conditional HSV-1 Mutants

The mutant HSV-1 vector, hrR3, was obtained from Dr. S. K. Weller(University of Connecticut Medical School) [Goldstein and Weller, JVirol 62: 2970-7, 1988]. It has an insertion of the E. coli lacZ geneinto the UL39 locus coding for the large subunit of ribonucleotidereductase/infected cell protein ICP6. LacZ expression is under controlof the ICP6 early virus promoter. Vector RR-P450, with an insertion ofthe cytochrome P450 gene into the lacZ locus of the hrR3 virus, wasprovided by Dr. E. A. Chiocca (Massachusetts General Hospital, Boston,Mass.).

HSV-1 vectors were grown on Vero cells (African green monkey kidneycells, ATCC #CCC81). Eighty to 90% confluent monolayers in 175 cm flaskswere infected at an MOI of 1 pfu/cell. At the time of maximal cytopathiceffect (˜36-48 h after infection), cells and supernatants were harvestedusing a cell scraper. Cells were lysed by 3 cycles of freezing andthawing, cell debris was spun down at 700 g for 10 min, and virus stockswere stored at −80° C. Virus titers were determined by standard plaqueassays [Roizman and Spear, J Virol 2, 83-84. 1968]. Virus stocks,typically 10¹⁰ plaque forming units (pfu)/ml, were thawed immediatelyprior to use. Procedures involving virus were performed in accordancewith the guidelines issued by the Harvard Office of Biological Safety.

The neural stem cells employed were derived from a stable, wellestablished, well studied, prototypical multipotent engraftable murineneural stem cell clone transfected with and constitutively expressingthe lacZ marker gene (clone C17.2) [Snyder et al., Cell 68: 33-51, 1992;Snyder and Macklis, Clin Neurosci 3: 310-16, 1996]. Described andcharacterized extensively elsewhere [Snyder, The Neuroscientist 4:408-25, 1998], this clone of neural stem cells has been shown to be aneffective vehicle for gene transfer to the CNS [Snyder et al., Nature374: 367-70, 1995; Lacorraza et al., Nature Med 4: 424-29, 1996]. TheCNS-1 rat glioma cell line was generated from a glioma induced in aLewis rat by treatment with N-nitroso-N-methylurea [Kruse et al., JNeuro-Oncology 22: 191-200, 1994] and was obtained from Drs. C. A. Kruseand W. F. Hickey (University of Colorado Health Sciences Center, Denver,Colo.). CNS-1 cells were grown as monolayers in Dulbecco's modifiedEagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100U/ml penicillin, and 100 μg/ml streptomycin (Gibco, Gaithersburg, Md.)at 37° C. in 5% carbon dioxide.

HSV-1 Amplicon Vectors

A series of amplicon plasmids were used to generate amplicon vectors:pHSV16, PHSVRR, and pHSVlacZ. To construct PHSV 16, a 1.9 kb fragmentcontaining the coding sequence of VP 16 was cut out from PNFT(ATCC#68668) with PstI and HindIII ligated into pHSVPrPUC (kindlyprovided by Dr. H. Federoff, University of Rochester, Rochester, N.Y.)after digestion with BamHI and partial digestion with PstI. In thisplasmid, VP 16 is driven by the HSV IE4/5 promoter. pHSVRR, expressingRR from an IE 4/5 promoter, was constructed by inserting a EcoR VlXtXhoI(partial digest) 4.8 kB fragment from pKHF (kindly provided by Dr. S.Weller, University of Connecticut) containing the coding sequence forboth the long subunit (UL39) and the short subunit (UL40) of ICP6/RRinto the SalIIBamHI (blunted) sites of the potylinker site of thepHSVPrPUC. pHSViacZ expresses lacZ under the control of the IE4/5promoter [Geller and Breakefield, Science 241, 1667-9, 1988].

Helper virus-free stocks of HSVRR, HSV 16, and HSVIacZ amplicons weregenerated according to the method developed by Fraefel et al. [J Virol70, 7190-7, 1996]. A set of 5 overlapping cosmids, which cover the wholeHSV-1 genome [Cunningham and Davison, Virologv 197, 116-24, 1993] andare deleted at their packaging signals, were kindly provided by Dr. C.Fraefel (Massachusetts General Hospital, Boston, Mass.). 106 Vero 2-2cells [Smith et al., 1992] were plated in p60 dishes and transfectedwith 0.6 μg plasmid DNA, as well as 0.2 μg DNA each of the 5 cosmidsusing LipofectAMINE (Gibco BRL, Life Technologies, Rockville, Md.).Using this method, only amplicon DNA is packaged into virions, as onlythis DNA contains a packaging signal. On day 3 after transfection,amplicon stocks were harvested by scraping the cells, three cycles offreezing and thawing, and sonication for 16 sec. Helper-free ampliconstocks were checked for the presence of wild type virus by infecting100,000 Vero cells per well in a 24-well plate with 500 41 ampliconstock per well and then observing for cytopathic effect. No ampliconstocks contained detectable wild type virus by this assay. HSV-IacZamplicon stocks were titered on confluent Vero cells by staining forP-galactosidase 16 h after infection for 4 hours at 37° C. in the X-galstaining solution containing 5 mM potassium ferrocyanide, 5 mM potassiumferricyanide, 2 mM magnesium chloride, 1 mg/ml X-gal (Fisher,Pittsburgh, Pa.) and 2.5% di-methyl-sulfoxide (Sigma, St. Louis, Mo.) inPBS [Turner et al., Stain Technol 65, 55-67, 1990].

Treatment of Neural Stem Cells with Mimosine and GCV, and InfectionTreated Neural Stem Cells with HSV-1 Mutants

Neural stem cells were grown in 24-well plates until they reachedconfluency (approximately 400,000 cells/well). Medium was changed everythird day. Starting at confluency, day 0, some wells were treated with400 μM mimosine (Sigma, St. Louis, Mo.). Medium was replaced on day 4and day 6. On day 6, some mimosine-pretreated wells were additionallytreated with 5 μM GCV (GCV, Cytovene-IV, Hoffmann La Roche, Nutley,N.J.). Cell culture medium was changed again on days 10, 13 and 17. Ondays 10 and 13 some previously mimosine ±GCV wells were washed 3 timeswith Hanks balanced medium (HBSS) and put back into growth medium. Inaddition, some mimosine ±GCV wells were washed, trypsinized, and split1:8. Cell morphology and cells/well for different treatment conditionswere determined in duplicate on days 0, 4, 7, 10, 13, and 17. The numberof cells/well was determined using a Coulter Counter (CoulterElectronics, Hialeah, Fla.).

To determine the effects of mimosine and GCV treatment on replication ofRR-HSV-1 mutants hrR3 and RRP450 in neural stem cells, cells werecounted in duplicate on day 7 of treatment and infected at an MOI of 1or 10. Virus titers in the conditioned medium—and in some experimentsalso in cell lysates—were determined by standard plaque tests on Verocells. Virus titers and nwnbers of viable cells were determined on days10, 13, and 17 after the start of mimosine treatment. Also, theinfluence of the expression of viral proteins VP 16 and ICP6 (RR),mediated by infection with amplicon vectors, on the replication of hrR3mutant virus was determined at different time points after infection ofneural stem cells. Stocks of helper-free packaged amplicons HSV 16.HSVRR and HSVIacZ (100 μl each) were added to the wells on day 7, 10, or13. Three days later titers of hrR3 in the medium were determined byplaque assay.

In Vivo Experiments and Histological Analysis

Intracerebral injection of CHS-1 tumor cells was carried out as follows:Female 8-10-week-old nude mice (randomly bred Swiss-White wereanesthetized by intraperitoneal (i.p.) injection with 70 μl of asolution consisting of 2 parts bacteriostatic 0.9% NaCl (Abbott, Ill.),and 1 part each of 20 mg/ml xylazine (Rompun™, Miles, Kans.) and 100mg/ml ketamine (Ketalar™, Parke-Davis, N.J.). After positioning theanimals in a stereotactic apparatus (Kopf, Tujunga, Calif.), a midlineskin incision was made, and a burr hole was drilled˜2 mm lateral and Imm anterior to bregma. Cells were injected into the forebrain over aperiod of 3-5 min to a depth of 2-4 mm from the dura using a Hamiltonsyringe. The needle was gradually retracted over 3-5 min, the burr holewas closed with bone wax (Ethicon). and the wound was washed withBetadine antiseptic (Purdue Frederick, Norwalk, Conn.). For secondaryinjections the same procedure was repeated.

For in vivo experiments, neural stem cells were grown to confluency in6-well plates. Treatment with 400 μM mimosine was started on day 0, andmedium was changed on days 4 and 7. On day 6, 5 μM GCV was added to somewells. On day 7, the cells per well were counted, and remaining wellswere infected with RR-P450 at an MOI of 1. On day 10, the medium andcell lysates of infected neural stem cells were assayed in a plaquetest; and the remaining wells were washed 3 times, trypsinized, counted,and resuspended in DMEM, or DMEM +5 μM GCV, at a concentration of 50,000cells/)il. Two μl aliquots of this cell suspension were injected intointracerebral gliomas in nude mice. The gliomas were produced byimplanting 5 days earlier an injection of 200,000 CNS-1 cells into theright frontal lobe. Also, in one experiment, 2 mice each were injectedwith 10⁴ or 10⁵ pfu RRP450 in 2 μl DMEM directly into the frontal lobetumor.

Intracerebral injection of CNS-1 tumor cells, virus and neural stemcells were carried out as follows: male 6-8 weeks old nu/nu nude mice(obtained from the MGH Facility/Edwin Steele Laboratory) wereanesthetized by intraperitoneal (i.p.) injection with 70 μl of asolution consisting of 2 parts bacteriostatic 0.9% NaCl (Abbott, Ill.),and 1 part each of 20 mg/ml xylazine (Rompun, Miles, K A) and 100 mg/mlketamine (Ketalar™, Parke-Davis, N.J.). After positioning the animals ina stereotactic apparatus (Kopf, Tujunga, Calif.), a midline skinincision was made, and a burr hole was drilled 2 mm rostral and 2 mmright of bregma. Cells were injected over a period of at least 2 min toa depth of 2.5 mm from the dura using a Hamilton syringe. The needle wasgradually retracted over 2 min, the burr hole was closed with bone wax(Ethicon), and the wound was washed with Betadine (Purdue Frederick,Norwalk, Conn.). For secondary injections the same procedure wasrepeated.

For histological analysis, animals were euthanized on day 13 (3 daysafter intracranial injection of virus-infected neural stem cells, virusor vehicle) by deep anesthesia with at least 500 pl ketamine/xylazinesolution i.p. and subsequent perfusion with a 4% paraformaldehydesolution in PBS (4% PFA). The brain was removed and post-fixed in 4% PFAfor 2 days, cryo-protected in 30% sucrose in PBS, and frozen at −80° C.Brains were sectioned at 12 μM using a cryostat and stained with neutralred. For lacZ-encoded β-galactosidase staining, mounted slices wereplaced for 24 hours at 37° C. in X-gal staining solution containing 5 mMpotassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM magnesiumchloride, 1 mg/ml X-gal (Fisher, Pittsburgh, Pa.), and 2.5%di-methylsulfoxide (Sigma, St. Louis, Mo.) in PBS (Turner et al., 1990,ibid). The sections were washed with PBS and counterstained withhematoxylin. Immunohistochemistry was performed using standardbiotin-avidin-bound peroxidase techniques (Vectastain ABC kit, Vector,Burlingame, Calif.) and a primary antibody against HSV-TK (rabbitpolyclonal, dilution 1:500, gift of Dr. W. Summers, Yale UniversitySchool of Medicine, New Haven, Conn.). Methylgreen was used as acounterstain. Some sections were double-stained for β-galactosidase andHSV-TK. In those cases, β-galactosidase staining was performed first,followed by HSV-TK immunohistochemistry and hematoxylin counterstaining.

Example 1 Migratory Capacity of NSCs in Culture

To determine properties of the NSCs in association with glioma cells,studies were initially performed in culture comparing the relativemigratory capacity of NS-, (clone C17.2) to fibroblasts (thelacZ-expressing TR-10 fibroblast cell line) when cocultured with gliomacells. C17.2 and TR-10 cells were maintained in Dulbecco's modifiedEagle's medium (DMEM; Mediatech, Washington, D.C.) supplemented with I0% fetal calf serum (FCS; Sigma, St. Louise, Mo.), 5% horse serum(HS;Gibco), 1% Glutamine (2 mM; Gibco), 1% penicillin/streptomycin(Sigma). CNS-1 cells were stably transduced with the PGK-GFP-IRES-NeoRretroviral vector construct to express green fluorescent protein (GFP)as previously described [Aboody-Guterman et. al, 1997], and maintainedin RPNH-1640 (Bio Whittaker) supplemented with 10% FCS and 1%penicillin/streptomycin (Sigrna). Cell structure studies were performedin 100 mm petri dishes under standard conditions: humidified, 37° C., 5%C02 incubator. CNS-1 glioma cells were plated to approx. 60-70%confluency around a 5 mm cylinder (i.e. free of CNS-1 cells) into which40,000 C17.2 or TR-10 cells plated overnight. At the same time, 40,000C17.2 or TR-10 cells were placed into a 5 mm cylinder placed directly ontop of adhered CNS-1 cells. The next day, the cylinders were removed andplates rinsed well with PBS to remove any floating cells, media wasreplaced, and plates incubated for 5 days. Plates were subsequentlystained for 0-galactosidase overnight after 5% glutaraldehyde fixation.(Note: both C17.2 and TR-10 cells are >90% blue with X-gal staining).

There was a wide distribution of C17.2 cells (FIG. 1B), suggesting thatthey had migrated far from their initial sites in the cylinder, comparedto the TR-10 cells (FIG. 1A), which remained localized to the area ofinitial seeding in the cylinders. These patterns were observed whetherthe cells were plated directly on top of the glioma cells (rightsidedcylinder arrows) or simply in juxtaposition to them (center cylinderarrows).

Example 2 Transgene-Expressing NSCs Migrate Throughout and BeyondInvading Tumor Mass in Vivo

To determine the behavior of clone C17.2 NSCs introduced into braintumors, experimental animals (syngeneic adult rats) first received animplant of 4×10⁴ D74 rat glioma cells in 1 μl injected into the rightfrontal lobe. Four days later, 1×10 ⁵ C17.2 NSCs in 1.5 μl PBS wereinjected at same coordinates directly into the D74 tumor bed. Animalswere then sacrificed at days 2, 6, and 10 days post-intratumoralinjection and cryostat sections of the brains were processed with X-galhistochemistry for P-galactosidase (β-gal) activity to detectdonor-derived cells and counterstained with neutral red to detect tumorcells. Donor C 1 7.2 NSCs were found extensively dispersed throughoutthe tumor within a few days, spanning an 8 mm width of tumor as rapidlyas 2 days after injection (FIGS. 2A, 2B). This is a much more extensiveand rapid dispersion compared to previous reports of 3T3 fibroblastsgrafted into an experimental brain tumor [Rainov et al., Cancer GeneTher 3: 99-106, 1996]. By day 10, C17.2 cells were seen throughout amajority of the tumor, clearly along the infiltrating tumor edge andslightly beyond it, drawn somewhat by the degenerative environment,seeming to “track” migrating tumor cells (FIGS. 2C, 2D). C17.2 cellsthemselves did not become tumorigenic. (FIG. 2A) Day 2 shown at 4X;arrowheads demarcate the approximate edges of tumor mass; even at lowerpower, the tumor can be seen to be intermixed with blue NSCs [arrows].This is appreciated more dramatically at high power in (FIG. 2B) at 10xwhere X-gal+, blue-staining NSCs (arrow) are interspersed between tumorcells staining dark red. (FIG. 2C) This view of the tumor mass, 10 daysafter intra-tumoral injection nicely shows that X-gal+blue, C17.2 NSCshave infiltrated the tumor but largely stop at the edge of the darklyred stained tumor tissue (border indicated by arrowheads) with somemigration into surrounding tissue when blue-staining NSC appears to be“following” and invading, “escaping” tumor cell (arrow) 10X. Thisphenomenon becomes even more dramatic when examining the behavior ofC17.2 NSCs in an even more virulent, invasive and aggressive tumor thanD74, the experimental CNS-I astrocytoma in the brain of a nude mouse(FIG. 2D). CNS-1 tumor cells were implanted into an adult nude mousefrontal cortex (day 0). On day 6, 4×10⁴ C17.2 cells were implanteddirectly into the tumor bed. The animal pictured in (FIG. 2D) wassacrificed on day 12 post-tumor implantation, 6 days post-intra-tumoralinjection. The cryostat section pictured was processed with X-galhistochemistry for β-galactosidase activity to detect blue C17.2 NSCsand counterstained with neutral red to show dark red tumor cells. Thereis extensive migration and distribution of blue C17.2 cells throughoutthe infiltrating experimental tumor bed, up to and along theinfiltrating tumor edge (white arrows), and, where many tumor cells areinvading normal tissue, into surrounding tissue in virtual juxtapositionto aggressive tumor cells (arrows) (10X).

Example 3 NSCs “Track” Infiltrating Tumor Cells

CNS-1 tumor cells were labeled by retroviral transduction with greenfluorescent protein (GFP), prior to implantation, to better distinguishsingle cells away from the main tumor bed [Aboody-Guterman et al.,NeuroReport 8: 3801-08, 1997]. GFP-expressing CNS-1 glioma cells (3×10⁴)in 1 μl PBS injected into right frontal lobe at stereotaxic coordinates2 mm lateral to bregma, on coronal suture, 3 mm depth from dura. 4×10⁴C17.2 or TR-10 cells in 1 μl PBS injected at same coordinates directlyinto tumor bed on day 6. 3-4 C17.2 animals (2 BUdR labeled, 1 BUdRpulsed) and 1-2 TR-10 control animals (1 BUdR labeled). Animals weresacrificed on days 9, 12, 16 and 21 post-tumor implantation. Cryostatsectioned, fixed brain tissue was stained either with 0-galactosidase(C17.2 cells blue) and neutral red (tumor cells dark red) or doubleimmunofluorescence with Texas Red anti-p-galactosidase (C17.2 cells red)and FITC anti-GFP (tumor cells green). FIGS. 3A, 3B show parallelsections: low power of C17.2 cells distributed throughout tumor andsurrounding edge (FIG. 3A) X-gal and neutral red (FIG. 3B) doubleimmunofluorescent labeling with Texas red and FITC is (FIGS. 3C, 3D) lowand high power of single migrating tumor cell in juxtaposition to C17.2cell (X-gal and neutral red) is FIGS. 3E and 3F show low and high powermagnification of single migrating tumor cell in juxtaposition to C17.2cell (X-gal and neutral red) (FIGS. 3G, 3H) low and high power of singlemigrating tumor cells in juxtaposition to C17.2 cells (doubleimmunofluorescent labeling with Texas Red and FITC).

Example 4 NSCs Implanted at Distant Site Migrate Toward Tumor

To examine the capacity of NSCs to migrate through normal tissue andspecifically target tumor cells, donor NSCs were injected intouninvolved sites distant from the main tumor bed in three separateparadigms, into the same hemisphere, into the opposite hemisphere, orinto the lateral ventricles.

Same hemisphere: CNS-1 glioma cells (3×10⁴) in 1 μl PBS was injectedinto the right frontal lobe at stereotaxic coordinates 2 mm lateral tobregma, on coronal suture, 3 mm depth from dura. 4×10⁴ C 17.2 or TR-10cells in 1 μl PBS injected into right frontal parietal lobe atstereotaxic coordinates 3 mm lateral and 4 mm caudal to bregma, 3 mmdepth from dura on day 6. Two animals were sacrificed at days 12 and 21.At all time points, NSCs were found distributed within the main tumorbed as well as in juxtaposition to migrating tumor cells in surroundingtissue (FIGS. 4A, 4B).

Opposite hemisphere: 3×10⁴ CNS-1 tumor cells in 1 μl PBS injected intoleft frontal lobe at stereotaxic coordinates 2 mm lateral to bregma, oncoronal suture, 3 mm depths from dura, 5×10⁴ CNS-1 tumor cells in 1 □lPBS injected into left frontoparietal lobe 3 mm lateral and 4 mm caudalto bregma, 3 mm depth from dura, 8×10⁴ C17.2 cells in 2 □l PBS injectedinto right frontal lobe 2 mm lateral and 2 mm caudal to bregma, 3 mmdepth from dura on day 6. Two animals sacrificed on day 12 and 21.(control—no tumor Coordinates: 2 mm R of bregma, 2 mm caudal, 3 mmdeep). NSCs were seen actively migrating across the central commissuretowards the tumor on the opposite side of the brain, and then enteringthe tumor (FIGS. 4C, 4D, 4E).

Implantation Away from CNS-1 Tumor Bed (Intraventricular):

In this final paradigm 5×10⁴ CNS-1 tumor cells in I ill PBS was injectedinto the right frontal lobe 2 mm lateral to bregma, on coronal suture, 3mm depth from dura. 8×10⁴ C17.2 cells in 2 μl PBS injected into left orright ventricle I mm lateral and 3 mm caudal to bregma, 2 mm depth fromdura on day 6. Two animals sacrificed on days 12 and 21. NSCs again wereseen within the main tumor bed, as well as in juxtaposition to migratingtumor cells (FIGS. 4F, 4G). In each case, donor NSCs were found tomigrate through normal tissue and “target” the tumor.

Example 5 Effect of Mimosine and GCV Treatment on Cell Number andMorphology of Neural Stem Ceils

To achieve improved transgene delivery to brain tumor cells infiltratingthe brain parenchyma, a novel cell-based delivery system forreplication-conditional HSV-1 vectors was developed. This employs neuralstem cells, which migrate throughout the tumor and beyond thetumor/parenchyma border, and ribonucleotide reductase-deficient HSV-1mutants, hrR3 and RRP450, which selectively replicate in dividing cells.Neural stems were infected with virus in culture where viral replicationcould be reversibly and completely abolished by treatment with mimosine,with reactivation upon removal of mimosine and cell division. Upon invivo injection of neural stem cells bearing quiescent virus intoestablished intracranial gliomas, virus replication was activated,presumably after some delay. Subsequently, foci of HSV-TK-positive tumorcells were found throughout the tumor and in the surrounding parenchyma.HSV-1-infected tumor cells appeared to be more widely distributed thanafter direct injection of the same HSV-1 mutant, suggesting an extensionof the range of HSV-1 vector delivery using this cell-based delayedactivation system.

Co-treatment with GCV as a viral replication block was also tested.After GCV treatment, neural stem cells differentiated into neurons andpotentially harbored the virus in a latent state. After withdrawal ofGCV and mimosine and splitting of the cultures on day 10, virus titersremained below levels of detection three and six days later. However,viral genomes were shown in infected cells by superinfection with helpervirus-free HSV-1 amplicon vectors carrying viral genes known to beinvolved in HSV-1 action, including the RR-gene and more effectively theRR and VP16 genes. Delivery of VP16 alone was insufficient forreactivation. Thus, it seems crucial to supply high levels of RRexpression to guarantee the reentry of the quiescent genome ofreplication-conditional RR-HSV into the replicative cycle.

Neural stem cell cultures were treated with mimosine (400 μM) and GCV (5μM) after reaching confluency. Cells/well remained at around 400,000cells for 4 and 7 days after the beginning of the mimosine treatment. Incontrast, untreated neural stem cell cultures continued proliferatingover the same period, reaching a density of 1.7×10⁶ cells/ml on day 10.Additional treatment with 5 μM GCV starting on day 6 after the beginningof mimosine treatment did not significantly affect cell viability, ascompared to treatment with mimosine alone. When mimosine alone ormimosine plus GCV were removed on day 10 after the beginning oftreatment, growth of neural stem cells resumed. Re-growth of neural stemcells occurred at a faster rate if cells were additionally rinsed andsplit 1:8 after removal of drug(s) on day 10. Re-growth of neural stemcells also occurred, if cells were kept on mimosine until day 13 andthen rinsed and split.

Mimosine-treated cultures of neural stem cells were infected withreplication-conditional HSV-1 mutants, hrR3 or RRP450, at an MOI of 1 onday 7 after the beginning of treatment. The genotype of hrR3 is RR-LacZ⁺ [Goldstein and Weller, Virology 166: 141-51, 1988] with the lacZgene under control of the ICP6 early viral promoter. RRP450 was derivedfrom hrR3 by replacement of the lacZ gene with the cytochrome P450 gene[Chase et al., Nat Biotechnol 16: 444-8, 1998]. Virus infection ofmimosine-arrested cells at an MOI=1.0 did not affect cell numbers; onday 10, three days after infection, cell numbers in infected anduninfected wells were not significantly different. Infected culturesthat were kept on mimosine treatment or mimosine plus GCV treatmentremained at the same cell number on day 13 and 17 after the start oftreatment. Infection with a higher MOI of 10, however, did lead to atypical cytopathic effect associated with virus replication inmimosine-treated cells, but not in mimosine plus GCV-treated cells.After removal of mimosine or mimosine plus GCV on day 10 and passagingof cultures at a ratio of 1:8, cell numbers increased less rapidly inthe mimosine-treated, as compared to the mimosine plus GCV-treated,cultures. Perhaps the mimosine only-treated cultures had more rapidcommencement of virus replication than the mimosine plus GCV-treatedcultures.

Treatment of neural stem cells with mimosine alone did not induce asignificant cytopathic effect. If cells at a density below confluencywere treated with mimosine 400 μM, they developed extensions and a moreneuronal shape. Additional treatment with GCV did not change thismorphology, nor did superinfection of mimosine-treated cultures withRRP450 (MOI=1.0); whereas, infection of untreated cultures virtuallywiped out all cells within a few days. Cultures which were previouslytreated with mimosine, infected on day 7, and split 1:8 into mediawithout mimosine on day 10 showed some cytopathic effect (CPE) on day13. In contrast, no CPE was seen in uninfected, mimosine-treated andsplit cultures, infected cultures that had been treated with mimosineand GCV from day 7-10, or mimosine-treated, infected cultures that werenot split. On day 17, non-split infected cultures previously treatedwith mimosine showed some CPE; whereas, mimosine-pretreated, infectedand split cultures showed extensive CPE on day 17. By day 17, low levelCPE was seen in cell cultures that had been infected and treated withmimosine and GCV. In summary, the presence of mimosine or mimosine plusGCV prevented virally induced CPE; whereas, the removal of these drugsinduced CPE. CPE due to virus propagation was greater in proliferatingneural stem cells.

Example 6 Effect of Mimosine With and Without GCVpre-Treatment on VirusTiters in the Medium of RRP450-Infected Neural Stem Cell Cultures

Sub-confluent cultures of neural stem cells were pretreated with 400 μMmimosine for 7 days and then infected with RRP450 at an MOI of 1 (4×10⁵pfu/well). Infectious medium was removed and fresh medium containingmimosine was added the day after infection. Some wells were additionallytreated with 5 μM GCV one day before infection, and GCV treatment wascontinued until at least day 10. On day 10, untreated, infected culturesshowed a marked cytopathic effect and high titers of virus in themedium, exceeding 10,000 pfu/ml (Table 1). FIG. 7 shows a growth curveof neural stem cells following treatment with mimosine alone or mimosineand GCV which were infected with RRP450 replication-conditional virus ata MOI 1 on day 7.

In contrast, treatment with mimosine and GCV completely abolished virusreplication on day 10. Removal of mimosine alone without splitting thecells did not induce detectable replication of RRP450, as measured bypfu in media (Table 1), although some CPE was seen in cells. Removal ofmimosine and GCV induced only low level virus replication, 1 out of 8cell lysates had virus titers of 200 pfu/400,000 cells on day 13 or 17(Table 1), and some CPE was noted in cultures. However, removal ofmimosine with additional splitting of cultures (1:8) induced notablevirus replication with some delay: Titers were very low—440 pfU/100,000cells— TABLE 1 Titers of RRP450 generated by untreated and drug-treatedneural stem cells. RRP450^(c) Day^(a) Treatment^(b) (pfu/100,000 cells)10 D10 >>10,000 Min <10 Min + GCV <10 13 Mim removal, not split <10 Mimremoval, split 1:8 440 +/− 280 Mim + GCV removal, <10 Split 1:8^(a)Treatment began on day 0. Mimosine and GCV treatment was removedfrom all cultures on day 10, and some cultures were split 1:8.^(b)Abbreviations: D10 = Dulbecco's modification of Eagle's medium + 10%fetal calf serum + antibiotics; Mim = 400 μM mimosine; GCV = 5 μM GCV.^(c)Cells were infected on day 7 with virus at MOI = 1. Media wereharvested on days 10, 13 and 17 after beginning of treatment and assayedvirus was titered by plaque assay on 2-2 cells.

3 days after passage (day 13), but increased greatly 7 days afterpassage (day 17) to 12000 pfu/100,000 cells, i.e. equivalent to 12% ofthe input virus titer. Thus, using confluent neural stem cells treatedwith mimosine or mimosine plus GCV, the replication of RRP450 could besuppressed for about 2 weeks and then reactivated in a delayed mannerupon removal of drug(s) and splitting of cultures. FIG. 5 shows theschedule of drug treatment and superinfection of neural stem cells withribonucleotide reductase-negative HSC-1 mutants. Early passages ofneural stem cells in log-growth phase were gown to confluency (400,000cells per well in 24-well dish), treated with 400 μM mimosine and 5 μMganciclovir (GCV), and infected with hrR3 or RRP450 at an MOI of 1.

Example 7 Stimulation of RR-Deficient Virus Replication inGCV/Mimosine-Treated Neural Stem Cells by Infection with AmpliconVectors Expressing lacZ, VP 16, and/or RR

To further analyze factors that stimulate reactivation of RR-negativeHSV-1 mutants following mimosine/GCV treatment of neural stem cells,cells infected according to the protocol, were subsequently infectedwith helper virus-free HSV-1 amplicon vectors expressing different virusproteins. Infection of hrR3-infected, growth arrested cells withamplicon vectors on day 7 yielded no titrable virus on day 10, sincemimosine and GCV were present during the entire time and blocked virusreplication. When mimosine and GCV were removed on day 10 and infectionwith amplicon vectors was carried out on day 10 or 13, only the vectorcoding for RR (HSVRR) induced reactivation whereas, vectors VP 16 (HSV16) and lacZ (HSVlacZ) and the no-vector control did not inducereplication in these non-dividing cells (Table 2). The induction ofreplication by superinfection with an amplicon vector expressing RR wasmuch higher when superinfection was done on day 10 as compared to day13. This suggests some loss of quiescent HSV-1 DNA during the time ofarrested virus replication. On day 10, co-infection with ampliconvectors coding for RR and VP16 markedly enhanced titers >50,000 pfu/ml,as compared to superinfection with RR-amplicon alone. Co-infection withlacZ-expressing amplicon vectors did not enhance titers (Table 2).

In another experiment, neural stem cell cultures treated with mimosineand GCV through day 10 and infected with hrR3 on day 7, were treated onday 10 with dexamethasone 10⁻ ⁷ M), which is known to be a potentinductor of HSV-1 reactivation; but virus titers in the media and celllysates remained below detection levels (10 pfu/100,000 cells).

FIG. 6 shows a growth curve of uninfected neural stem cells followingtreatment with mimosine alone or mimosine and GCV and/or infection withreplication-conditional virus. Mim means cells treated with 400 μMmimosine; MG means cells treated with 400 μM mimosine and 5 μM GCV;split means cell cultures split 1:8 on day 10.

Example 8 In Vivo Experiments

One hundred thousand control neural stem cells or cells treated withmimosine plus GCV and infected with RRP450 (MOI 1) were injected on day10 directly into intracranial CNS-1 gliomas in nude mice. The CNS-1gliomas had been established by implanting 200,000 CNS-1 cells into theright frontal lobe five days before neural stem cell treatment.Histology of tumor and surrounding brain parenchyma was performed on day3 and 6 after injection of neural stem cells. TABLE 2 Titers of hrR3generated by superinfection of hrR3-infected and drug-treated neuralstem cell cultures with helper-free packaged amplicon vectors. AmpliconHrR3^(c) Day^(a) Vector^(b) titers (pfu/100,000 cells) 7 no vector 0HSVRR 0 HSV16 0 HSV16 + HSVRR 0 HSVlacZ + HSVRR 0 10 no vector 0 HSVRR2400 +/− 900  HSV16 0 HSV16 + HSVRR >50000 HSVlacZ 0 HSVlacZ + HSVRR2300 +/− 1155 13 no vector 0 HSVRR 15 +/− 5  HSV16 0 HSV16 + HSVRR 40+/− 10 HSVlacZ 0 HSVlacZ + HSVRR 20^(a)Day of infection with amplicon vector. On day 7 neural system cellstreated with mimosine and GCV were infected with hrR3 at MOI 1. On day10 mimosine and GCV were removed. On days 7, 10, and 13, cultures wereinfected with 100 μl helper-virus free HSV-1 amplicon vectors carryingthe genes for RR (HSVRR), VP16 (HSV 16), or lacZ (HSVlacZ).^(b)HSVRR amplicon construct expresses ribonucleotide reductase undercontrol of the IE4/5 promoter; HSV16 amplicon expresses VP16 under theIE4/5 promoter; and HSV lacZ amplicon expresses E. coli lacZ under theIE4/5 promoter.^(c)By standard placque assays, the titer of hrR3 in the media wasdetermined 3 days after superinfection with amplicon vectors or vehicle.“0” is shown if the count was below detection level (<10 pfu/100,000cells).

At both time points, β-galactosidase staining showed many positive cellsin both control and treated neural stem cells throughout the wholeglioma. β-galactosidase-positive single cells were also observedinvading the CNS parenchyma around the glioma. However, consistent withdata in culture the activation of replication of RRP450 in infectedcells, as detected by immunohistochemistry of HSV-TK, was low. Only veryfew HSV-TK positive cells were found on day 3 and 6 after intra-tumoralinjection. On sections double stained for HSV-TK and β-galactosidase,most TK-positive cells within the tumor mass were β-galactosidasenegative, and thus presumably CNS-1 glioma cells infected with virusreleased by neural stem cells.

Some neural stem cell cultures had also been treated with mimosinealone, without GCV, and infected with RRP450 at an MOI of 1. Three daysafter infection, 10⁵ neural stem cells were injected into pre-existingintracranial CNS-1 tumors. Histology was analyzed three days later.Staining with β-galactosidase showed that neural stem cells had migratedthroughout the tumor, as described above for the mimosine- andGCV-pretreated cells. However, in this experiment the number of lacZ+neural stem cells was greatly reduced, as compared to the same number ofneural stem cells which had been infected with hrR3 and treated withboth mimosine and GCV. The disappearance of lacZ+ neural stem cells canbe explained by analogy with experiments in culture in whichreactivation and replication of the RRP450 mutant virus subsequentlykilled host cells. In support of that proposal, HSV-TKimmunohistochemistry, used to mark cells actively replicating RRP450,revealed foci of reactivation throughout the glioma. The RRP450 virusdelivered by neural stem cells spread not only within the tumor, butalso into the brain parenchyma, where single HSV-TK positive cells werefound at some distance from the tumor/parenchyma border. SuchHSV-TK-positive cells might be single infiltrating neural stem cells inthe process of virus reactivation, infected glioma cells, or reactiveglial or immune cells. Presumably the TK-positive cells were notneurons, as the RRP450 virus cannot replicate in postmitotic cells.

The efficiency of HSV-TK gene delivery using the neural stem cell/RRP450delivery system was compared to standard direct injection of 10⁵ pfu ofthis same virus into an established CNS-1 intracranial tumor. Thisamount of virus is the same as used to infect the neural stem cellsprior to their intratumoral injection and is a more than 100 fold higherthan the titer of virus detected in the supernatant of mimosine-treated,RRP450-infected neural stem cells at the time of intracerebral injection(three days after infection, Table 1). Three days after direct injectionof the virus, immunohistochemistry showed that HSV-TK+ cells weredistributed throughout the tumor. However, after direct virus injection,HSV-TK+ cells were only rarely found beyond the brain/tumor border. Thiscontrasted with gene delivery by the neural stem cell/RRP450 system,following which single HSV-TK+ cells were found some distance outsidethe tumor/parenchyma border.

1. An isolated pluripotent neuronal cell having the capacity todifferentiate into at least different types of nerve cells, said cellbeing further characterized by a) having a migratory capacity wherebythe cell is capable of traveling from a first location where theneuronal cell is administered to a second location at which there is atleast one tumor cell; b) having the ability to travel through and arounda tumor, whereby a plurality of the neuronal cells are capable ofsurrounding the tumor; and c) having the capacity to track at least oneinfiltrating tumor cell, thereby treating infiltrating and metastasizingtumors.
 2. The neuronal cell of claim 1 wherein the neuronal cellcomprises an isolated neural stem cell.
 3. The neuronal cell of claim 1wherein the neuronal cell has been treated to secrete a cytotoxicsubstance.
 4. The neuronal cell of claim 1 wherein the neuronal cell hasbeen transformed with factors that directly promote differentiation ofneoplastic cells.
 5. The neuronal cell of claim 1 wherein the neuronalcell has been transformed with viral vectors encoding therapeutic genesto be incorporated by tumor cells.
 6. The neuronal cell of claim 1wherein the neuronal cell has been transformed with viral vectorsencoding suicide genes, differentiating agents, or receptors to trophinsto be incorporated into tumor cells.
 7. The neuronal cell of claim 1wherein the neuronal cells administered on the same side or acontralateral side of the brain from the tumor are capable of reachingthe tumor.
 8. A method of converting a migrating neuronal cell to amigrating packaging/producer cell, said method comprising a) providing aneuronal cell which constitutively produces a marker such as β-gal; b)cotransfecting the neuronal cell with an amphotropic pPAM3 packagingplasmid and a puromycin selection plasmid pPGKpuro; c) selectingtransfected cells in puromycin; d) selecting for cell surface expressionof the amphotropic envelope glycoprotein coat; e) isolating cells byfluorescent activated cell sorting using monoclonal antibody 83A25; f)screening the cells of step e) for their packaging ability by assessingwhich colonies packaged lacZ into infectious viral particles; therebyproducing a migratory neuronal cell capable of being transfected with agene of choice, so that viral particles expressing the gene of choiceare produced and disseminated over a wide area of the central nervoussystem by a plurality of the transfected packaging cells.
 9. The methodof 8, wherein step f) is performed by a virus focus assay for β-galproduction.
 10. The method of 8, wherein the gene of choice is a prodrugactivation enzyme.
 11. The method of claim 10, wherein the prodrugactivation enzyme is E. coli cytosine deaminase (CD), HSV-TK orcytochrome p450.
 12. The method of claim 10, wherein the prodrugactivation enzyme is E. coli cytosine deaminase (CD).
 13. A novel cellpackaging line for the central nervous system, said cell line comprisingneuronal cells which constitutively produce a marker such as β-gal, theneuronal cells having been cotmnsfected with an amphotropic pPAM3packaging plasmid and a puromycin selection plasmid pPGKpuro; thetransfected cell being selected in puromycin, for cell surfaceexpression of the amphotropic envelope glycoprotein coat and forfluorescence using monoclonal antibody 83A25, and for their packagingability by assessing which colonies packaged lacZ into infectious viralparticles; the resulting cells being capable of packaging and releasingparticles or vectors which, in turn, may serve as vectors for genetransfer to central nervous system cells.
 14. The novel cell packagingline of claim 13, wherein the particles are replication-defectiveretroviral particles.
 15. The novel cell packaging fine of claim 13,wherein the vectors comprise replication-conditional herpes virusvectors.
 16. A neuronal stem cell comprising a vector encoding atherapeutic agent.
 17. The neuronal stem cell of claim 16, wherein thevector is a replication conditional vector.
 18. The neuronal stem cellof claim 16, wherein the vector is a herpes simplex vector.
 19. Theneuronal stem cell of claim 18, wherein the herpes simplex vector is aherpes simples type 1 vector.
 20. The neuronal stem cell of claim 19,wherein the herpes simplex type 1 vector is deficient for ribonucleotidereductase.
 21. A method of treating a brain tumor in a mammal in needthereof, said method comprising: a) providing a neuronal stem cellcomprising a vector encoding a therapeutic agent; and b) administeringsaid neuronal stem cell in a pharmaceutically acceptable carrier into amammal in need thereof.
 22. The method of claim 21, wherein the braintumor is a malignant glioma.
 23. The method of claim 21, wherein thevector is deficient for a component necessary for vector replication.24. The method of claim 23, wherein the component necessary for vectorreplication is ribonucleotide reductase.
 25. A method of treating abrain tumor in a mammal in need thereof said method comprising: a)providing a neuronal stem cell comprising a replication conditionalvector encoding a therapeutic agent; b) inhibiting replication of saidreplication conditional vector in said neuronal stem cell; c)administering the neuronal stem cell of step b in a pharmaceuticallyacceptable carrier into a mammal in need thereof; and, d) enhancingreplication of said replication conditional vector.
 26. The method ofclaim 25, wherein step b) is performed by inhibiting growth of neuronalstem cell.
 27. The method of claim 26, wherein growth inhibition isperformed using mimosine.
 28. The method of claim 26, wherein growthinhibition is performed using a combination of mimosine and ganciclovir.29. A method of treating a brain tumor in a mammal in need thereof saidmethod comprising: a. administering into a mammal a neuronal stem cellcomprising a herpes simplex type 1 vector encoding thymidine kinase; andb. administering ganciclovir into said mammal.
 30. A method of preparingneural stem cells encoding a therapeutic agent, said method comprising:a) providing a neural stem cell; b) growing said neural stem cell toconfluency; c) subjecting the neural stem cell to areplication-arresting protocol; d) infecting the replication arrestedcell with RR-P450; and e) washing the infected cell, separating the cellfrom its growth surface and resuspending the cell in a medium to obtaina concentration of 50,000 cells/μl.
 31. A method of preparing neuralstem cells encoding a therapeutic agent, said method comprising: a)providing a neural stem cell; b) growing said neural stem cell toconfluency; c) subjecting the neural stem cell to areplication-arresting protocol, said protocol comprising treating cellswith a medium comprising about 400 μM mimosine on days 0 and 4 andtreating cells on day 6 with a medium comprising about 400 μM mimosineand optionally about 5 μM ganciclovir; d) infecting the replicationarrested cell with RR-P450 at an MOI of 1 on day 7; and e) washing theinfected cell, trypsinizing and resuspending in DMEM and optionally 5 μMGCV to obtain a concentration of 50,000 cells/μl.